The objectives of this proposal are to elucidate the regulation of endogenous gamma-secretase and to understand the mechanism underlying Alzheimer's disease (AD) as well as to provide a molecular basis of drug development. gamma-Secretase as a drug target for Alzheimer's disease and cancer has been extensively investigated. Most of knowledge today on gamma-secretase mainly comes from studies that used an exogenous expression of one or more of the components of gamma-secretase in cellular models. Whether distinct gamma-secretase complexes and substrate specificity are associated with cell types, and possibly cell lineage and tissues is poorly understood. The proposed studies will investigate the regulation and function of the endogenous gamma-secretase complexes. The first aim of this application is to elucidate the mechanism of gamma-secretase activity and specificity in epithelial, hematopoietic neuronal origins. The second specific aim is to examine the shape of the active site within the gamma-secretase complex and determine the stoichiometry of the gamma-secretase complexes isolated from epithelial, hematopoietic and neuronal cell lines. Lastly, the third aim is to determine the mechanism and specificity of gamma-secretase activity in the PS1 mutation knock-in and analyze active site structure and complex composition. The proposed studies will develop a comprehensive understanding of endogenous gamma-secretase and provide insights into the structure and function of gamma-secretase and may lead to a better understanding of gamma-secretase in AD and cancer as well as the development more effective treatment for AD, malignancies and other human disorders.